Glycosaminoglycans (GAGs) are linear, acidic polysaccharides that exist ubiquitously in nature as residents of the extracellular matrix and at the cell surface of many different organisms of divergent phylogeny [Habuchi, O. (2000) Biochim Biophys Acta 1474, 115-27; Sasisekharan, R., Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) Proc Natl Acad Sci USA 90, 3660-4]. In addition to a structural role, GAGs act as critical modulators of a number of biochemical signaling events [Tumova, S., Woods, A., and Couchman, J. R. (2000) Int J Biochem Cell Biol 32, 269-88] requisite for cell growth and differentiation, cell adhesion and migration, and tissue morphogenesis.
Heparan sulfate like glycosaminoglycans (GAGS or HSGAGs) are present both at the cell surface and in the extracellular matrix. Heparin-like glycosaminoglycans are important components of the extracellular matrix that are believed to regulate a wide variety of cellular activities including invasion, migration, proliferation and adhesion (Khodapkar, et al. 1998; Woods, et al., 1998). HSGAGs accomplish some of these functions by binding to and regulating the biological activities of diverse molecules, including growth factors, morphogens, enzymes, extracellular proteins. HSGAGs are a group of complex polysaccharides that are variable in length, consisting of a disaccharide repeat unit composed of glucosamine and an uronic acid (either iduronic or glucuronic acid). The high degree of complexity for HSGAGs arises not only from their polydispersity and the possibility of two different uronic acid components, but also from differential modification at four positions of the disaccharide unit. Three positions, viz., C2 of the uronic acid and the C3, C6 positions of the glucosamine can be O-sulfated. In addition, C2 of the glucosamine can be N-acetylated or N-sulfated. Together, these modifications could theoretically lead to 32 possible disaccharide units, making HSGAGs potentially more information dense than either DNA (4 bases) or proteins (20 amino acids). It is this enormity of possible structural variants that allows HSGAGs to be involved in a large number of diverse biological processes, including angiogenesis (Sasisekharan, R., Moses, M. A., Nugent, M. A., Cooney, C. L. & Langer, R. (1994) Proc Natl Acad Sci USA, 1524-8.), embryogenesis (Binari, R. et al (1997) Development, 2623-32; Tsuda, M., et al. (1999) Nature, 276-80.; and Lin, X., et al (1999) Development, 3715-23.) and the formation of β-fibrils in Alzheimer's disease (McLaurin, J., et al (1999) Eur J Biochem, 1101-10. and Lindahl, B., et al (1999) J Biol Chem, 30631-5).
One specific example of an HSGAG is heparin. Heparin, a highly sulphated HSGAG produced by mast cells, is a widely used clinical anticoagulant, and is one of the first biopolymeric drugs and one of the few carbohydrate drugs. Heparin primarily elicits its effect through two mechanisms, both of which involve binding of antithrombin III (AT-III) to a specific pentasaccharide sequence, HNAc/S,6SGHNS,3S,6SI2SHNS,6S contained within the polymer. HSGAGs have also emerged as key players in a range of biological processes that range from angiogenesis [Folkman, J., Taylor, S., and Spillberg, C. (1983) Ciba Found Symp 100, 132-49] and cancer biology [Blackhall, F. H., Merry, C. L., Davies, E. J., and Jayson, G. C. (2001) Br J Cancer 85, 1094-8] to microbial pathogenesis [Shukla, et al (1999) Cell 99, 13-22]. HSGAGs have also recently been shown to play a fundamental role in multiple aspects of development [Perrimon, N. and Bernfield, M. (2000) Nature 404, 725-8]. The ability of HSGAGs to orchestrate multiple biological events is again likely a consequence of its structural complexity and information density [Sasisekharan, R. and Venkataraman, G. (2000) Curr Opin Chem Biol 4, 626-31].
Although the structure and chemistry of HSGAGs are fairly well understood, information on how specific HSGAG sequences modulate different biological processes has proven harder to obtain. Determination of these HSGAG sequence has been technically challenging. HSGAGs are naturally present in very limited quantities, which, unlike other biopolymers such as proteins and nucleic acids, cannot be readily amplified. Second, due to their highly charged character and structural heterogeneity, HSGAGs are not easily isolated from biological sources in a highly purified state. Additionally, the lack of sequence-specific tools to cleave HSGAGs in a manner analogous to DNA sequencing or restriction mapping has made sequencing a challenge.
Recently, in an effort to develop an understanding of HSGAG structure, focus has been placed on the cloning and characterization of the enzymes involved in HSGAG biosynthesis. Another, strategy for elucidating the structure of HSGAGs has been to employ specific HSGAG degradation procedures, including chemical or enzymatic cleavage, in conjunction with analytical methodologies, including gel electrophoresis or HPLC, to sequence HSGAGs. Recently, we have introduced a sequencing procedure that couples a bioinformatics framework with mass spectrometric and capillary electrophoretic procedures to sequence rapidly biologically important HSGAGs, including saccharide sequences involved in modulating anticoagulation. The sequencing methodology uses chemical and enzymatic tools to modify or degrade an unknown glycosaminoglycan polymer in a sequence-specific manner. (Venkataraman, G., et al., Science, 286, 537-542 (1999), and U.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000, having common inventorship).